The production of complementary DNA (cDNA) molecules from messenger RNA (mRNA) has allowed the isolation, characterization and expression of a large number of genetic sequences. The use of cDNA permits the production of higher eukaryotic proteins in microbial hosts, and is therefore central to the genetic engineering industry.
Numerous methods for producing cDNA have been described. For a general discussion of cDNA cloning methods, see Maniatis et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1983). In general, the common features of cDNA cloning strategies include isolation of mRNA, annealing of the mRNA to relatively short DNA primers followed by enzymatic synthesis of the first and second strands of DNA. The resulting double stranded molecules are inserted into a plasmid or bacteriophage vector and transformed into a host cell, generally the bacterium E. coli, to produce a "library" of cDNA molecules. The library is then screened by a variety of methods to identify clones of interest.
Early methods for producing cDNA were not well suited to cloning sequences corresponding to low-abundance messages, and often yielded incomplete clones. These methods relied on the use of the first cDNA strand as a primer for second strand synthesis, resulting in the formation of a hairpin loop which had to be removed by nuclease digestion. This digestion, together with premature termination of first strand synthesis, resulted in the cloning of incomplete cDNAs. Cloning of the entire coding sequence for a rare protein could therefore be extremely laborious, if not impossible, requiring many rounds of cloning and screening, then joining of cloned fragments to generate the complete sequence.
More recently, a number of methods have been described which produce a higher yield of full-length clones and are more suited to the cloning of rare sequences. These methods rely on enrichment for desired sequences or priming strategies which eliminate the need for nuclease treatment. Despite the advances represented by these methods, certain problems remain, including difficulty in cloning rare sequences that encode proteins known only by their intrinsic activities, which may require full-length, expressible cDNAs.
The need to efficiently express cloned sequences in order to identify clones of interest has led to the development of directional cloning techniques, whereby cDNA sequences are inserted into cloning vectors in a predetermined orientation. By providing transcriptional promoter and terminator sequences adjacent to the insertion site in the cloning vector, cloned sequences can be expressed, permitting screening of clones by detection of the gene product of interest. These directional cloning methods generally rely on the use of vector and cDNA sequences constructed with specific, complementary termini to obtain the desired insertion. Okayama and Berg (Mol. Cell. Biol. 2: 161-170, 1982) describe a cloning method in which a dT-tailed plasmid serves as a primer for first strand cDNA synthesis, and a primer-adapter is used for second strand synthesis. Shortcomings of this system include its complexity, the difficulty of preparing the vector components, and the inability to easily size fractionate the cDNA. The complexity of the system is evidenced by the numerous steps required to synthesize the cDNA and introduce the primer-adapter into the cDNA-vector construct. Preparation of the vector components is a multi-step process involving two physical separations, gel electrophoresis and affinity chromatography, which cause large losses of material, therefore necessitating the need for large starting amounts of vector and the use of large amounts of reagents. Even under optimum conditions, a fair amount of less then full-length cDNA is synthesized. The method of Coleclough et al. (Gene 34: 305-314, 1985) utilizes primer-adapters which are complementary to the ends of the cloning vector. Preparation of the vector requires a number of manipulative steps, and second strand cDNA synthesis is completed on the vector-cDNA complex. With such a complex, it is difficult to reproducibly carry out second strand synthesis with good efficiencies of cloning. Schmid et al. (Nuc. Acids Res. 15: 3987-3996, 1987) disclose a method utilizing specific primers to produce restriction site sticky ends on the cDNA insert. However, primer design is based on foreknowledge of the target sequence, limiting this method to the cloning of previously cloned DNAs. The method of Meissner et al. (Proc. Natl. Acad. Sci. USA 84: 4171, 1987) requires endonuclease digestion of the double-stranded cDNA to allow insertion into the vector. Sequences containing corresponding internal endonuclease cleavage sites would therefore be cleaved. As with the method of Coleclough et al., the method of Han et al. (Biochemistry 26: 1617-1625, 1987) requires synthesis of second strand cDNA on a complex of vector, cDNA, and adapter-primer. As previously mentioned, these are difficult procedures and therefore cloning efficiencies are extremely variable.
Most of the above-mentioned cloning methods do not allow amplification of the cloned cDNA by the polymerase chain reaction (PCR) (Scharf et al., Science 233: 1076, 1986; Saiki et al., Science 239: 487, 1988), which requires the presence of two primers in high concentration to initiate repeated rounds of DNA synthesis between the sites where the primers anneal. This is not possible in those procedures which require vector priming or synthesis of the second strand on a cDNA vector complex. Although the methods that use oligonucleotide primers could utilize PCR to amplify cDNA prior to attaching the cDNA to a cloning vector, these approaches are still limited by the need for endonuclease digestion of the cDNA prior to ligation to the cloning vector, and therefore carry the risk of digesting the coding sequence of the cDNA.
Consequently, there remains a need in the art for a cDNA cloning method that permits directional cloning of low-abundance messages at high efficiency, can further incorporate the use of PCR, is amenable to size fractionation of cDNA, obviates the use of endonucleases to tailor the cDNA for attachment to the cloning vector, and permits expression of the cDNA. The present invention provides such methods, as well as other, related advantages.